Classic immunological approaches to detection of proteins using antibodies, receptors, or other binding partners include, among others, enzyme-linked immunosorbent (ELISA) assay (generally in the form of an antibody sandwich method), radioimmunoassays, and immunoblotting methods (Burnette 1981, Engvall at al. 1971, and Yalow et al. 19601, with equivalent biochemical approaches being used for protein-receptor and protein-ligand reactions. Majority of these methods rely on examining one protein at a time. Moreover, the protein target typically must be present in large amounts and at relatively high concentrations to assure a reliably detectable signal.
Beginning in 1975 but largely over the last 15 years, methods that attempt to simultaneously examine many different proteins have appeared. These include 2-D gel electrophoresis, tandem mass spectrometry (MS-2 or MS-3) systems with intermediate protein cleavage, isotope-coded affinity tag (ICAT)-MS, MudPIT (LC-2/MS-2), and combinations of these approaches [Guerrera et al. 2005, Gygi et al. 1999, Klose 1975, and O'Farrell 1975]. For instance, in the ICAT approach, proteins from different tissues are labeled with tags containing either hydrogen or deuterium, and the differential patterns are observed by mass spectrometry. While many of these methods allow multiple samples to be compared concurrently, due to the cost of associated technologies such as mass spectroscopy, many of these approaches have not found general utility.
A relatively recent addition to the repertoire, protein arrays, in which fluorescently labeled proteins are allowed to bind to numerous spots, each containing covalently attached antibodies for a specific protein (antigen), are an appealing solution, as they can be mass produced and data analysis standardized [Angenendt 2005, Bussow at al. 1998, Cahill 2001, de Wildt at al. 2000]. While limited by the number of available specific antibodies that can function on a solid phase, a more important shortcoming of this method is the relative binding ability of the antibodies. Unlike DNA probes on gene expression microarrays, where probes can be selected to be fairly uniform in their binding affinity for mRNA targets, different antibodies may bind their fluorescently labeled antigens with very different affinities. Because of this variable affinity, quantification from spot to spot (antigen to antigen) becomes difficult, especially when combined with the likelihood that the fluorescent signal can lie outside of the linear range of detection. For example, low copy number proteins in the sample will not be seen, unless their binding is stronger than the average antigen-antibody interaction elsewhere on the chip, in which case they will be over-represented. Moreover, the effective concentration range will have the same floors and ceilings as other fluorescent methods on microarrays, and small changes in protein levels will be difficult to distinguish using protein arrays. Another common issue with fluorescent labeling is the existence of overlapping emission spectra, which limits the number of differentially labeled samples that can be applied to the arrays.
In summary, gene regulation analysis at the level of protein synthesis, like proteomics in general, lags behind nucleic acid analysis in its throughput, sensitivity and automation. This is due to the relatively poor stability of proteins, their high heterogeneity, and the requirement for a much wider dynamic range of detection with increased demand for sensitivity approaching the single molecule detection level, a need not easily met by fluorescent or colorimetric measurements. While protein arrays based on antibody interactions with fluorescently labeled antigens or secondary antibodies have gained some degree of popularity over the last decade [Cahill 2001], some of the drawbacks of this approach, including fluorescence saturation and overlap in fluorescent emission, make accurate quantification difficult. While there are currently several examples of application of nanopore-based analytics, no existing technology allows quantification of protein-protein interactions with a plurality of tags.